Use of glutamine synthetase for treating hyperammonemia

ABSTRACT

The present invention relates to the use of glutamine synthetase as a protein therapy (such as enzyme replacement protein therapy) for the treatment of hyperammonemia. In particular the invention relates to the systemic administration of glutamine synthetase. The glutamine synthetase may be provided in conjugated or fusion form, to increase its half-life in the circulation. Also provided is a pharmaceutical composition comprising glutamine synthetase. The invention also relates to the uses, methods and compositions involving a combination of the glutamine synthetase protein and an ammonia lowering agent, such as a nitrogen scavenger.

The present invention relates to the use of glutamine synthetase as aprotein therapy (such as enzyme replacement protein therapy) for thetreatment of hyperammonemia, and in particular to the systemicadministration of glutamine synthetase. The glutamine synthetase is thusadministered as a protein, or polypeptide, with the aim of increasingcirculating levels of glutamine synthetase. The glutamine synthetase maybe provided in conjugated or fusion form, to increase its half-life inthe circulation. Also provided is a pharmaceutical compositioncomprising glutamine synthetase. The invention also relates to the uses,methods and compositions involving a combination of the glutaminesynthetase protein and an ammonia lowering agent, such as a nitrogenscavenger.

Hyperammonemia is a metabolic condition characterised by increased, orexcess, ammonia in the blood. It is a dangerous condition, principallysince it may lead to increased entry of ammonia to the brain, which inturn causes neurological disorders and neuropsychiatric abnormalities,which can be very severe, leading to, inter alia, brain damage,seizures, retardation, coma and even death. Indeed, encephalopathy is acommon and hazardous complication of hyperammonemia. Although precisemechanisms for encephalopathy/brain damage are not yet understood,astrocytic osmotic stress caused by the increased ammonia is believed toplay a role, leading to cerebral oedema and increased intracranialpressure. Hyperammonemia may be congenital or acquired, and may beprimary or secondary.

Primary (congenital) hyperammonemia arises from various inborn errors ofmetabolism characterised by reduced activity of any of the enzymes ortransporter proteins of the urea cycle. Indeed, such inborn errors ofmetabolism form a group of diseases called urea cycle disorders (UCD).Ammonia (NH₃), which may co-exist in the body with its charged formammonium (NH₄ ⁺) depending on pH, is a product of the catabolism ofproteins and other nitrogenous compounds. It is converted by the enzymesof the urea cycle to the less toxic substance urea prior to excretion inurine by the kidneys. The urea cycle, which also functions as the solesource of production of certain amino acids (arginine, citrulline andornithine) in the body, comprises 6 enzymes, the five catalytic enzymescarbamoyl phosphate synthetase I (CPS1), ornithinetranscarbamylase(OTC), or argininosuccinic acid synthetase (ASS1), arginosuccinic acidlyase (ASL) and arginase (ARG), and the co-factor producing enzymeN-acetylglutamate synthetase (NAGS) and 2 transporter proteins ornithinetranslocase (ORNT1) and citrin. A defect can occur in any one or more ofthese 8 proteins. Severe deficiency or total absence of activity of anyof the first four enzymes or of NAGS result in accumulation of ammoniaand other precursor metabolites during the first few days of life.Infants with a severe UCD are normal at birth but rapidly developcerebral oedema and the related signs of lethargy, anorexia, hyper- orhypoventilation, hyperthermia, seizures, neurologic posturing and coma.Severity of the UCD is influenced by the position of the defectiveenzyme in the pathway and severity of the enzyme defect. With rapididentification and current treatment strategies, survival of neonateswith hyperammonemia has improved dramatically in the last few decades,but intellectual ability is typically impaired. In milder, or partial,deficiencies of these enzymes and in ARG deficiency ammonia accumulationmay be triggered by illness or stress at almost any time of life. Inthese disorders the elevations of plasma ammonia concentration andsymptoms are often more subtle than the neonatal presentation of a UCD,and the first recognised clinical episode may not occur for months ordecades.

Secondary hyperammonemia is caused by inborn errors of intermediarymetabolism characterised by reduced activity in enzymes/proteins thatare not part of the urea cycle, e.g. propionic acidaemia, methyl malonicacidaemia, galactosaemia, fatty acid oxidation disorders andmitochondrial disorders, or by dysfunction of cells (e.g. the liver)that make major contributions to ammonia metabolism and/or nitrogenmetabolism more generally.

Acquired hyperammonemia is usually caused by liver diseases, includingboth acute and chronic liver failure, such as viral hepatitis orexcessive alcohol consumption. Impaired liver function or vascularbypass of the liver, resulting in decreased filtration of blood in theliver, leads to hyperammonemia. Hepatic encephalopathy due tohyperammonemia is a common complication of liver disease.

Hyperammonemia may also occur for other reasons including renaldysfunction e.g. kidney dysfunction and/or failure, drug toxicity e.g.due to valproic acid or cyclophosphamide, idiopathic hyperammonemiasyndrome after immunosuppression or cytotoxic therapy, ureolysis instagnant urine and urinary tract infections, or essential amino acidtotal parenteral nutrition.

Current treatments for hyperammonemia are designed to reduce ammonialevels in the blood and/or the brain e.g. by haemodialysis (typicallyused in neonates) or by administering compounds that increase removal ofnitrogen waste, for example non-absorbable disaccharides (e.g.lactulose) or antibiotics (e.g. rifaxamin) or compounds that convertnitrogen into products other than urea which are then excreted, e.g.compounds such as sodium benzoate, arginine, carglumic acid,phenylacetate or more latterly phenylbutyrate, or L-ornithineL-aspartate (LOLA) or L-ornithine phenylacetate (OP).

Management of the condition may also include dietary control to restrictprotein intake and ensure adequate nutritional intake, includingmanagement of protein and/or nitrogen intake and parenteral intake ofcalories.

However, despite improvements in treatment and management of thecondition current therapies are non-specific and do not always succeedin managing the condition successfully. In the case of UCD particularly,current therapies may not prevent many elevated ammonia events andpatients with severe forms of the disease are often assessed for livertransplant around age 5. There is therefore a continuing need forfurther or improved therapies for hyperammonemia.

The present invention seeks to address this need and is based on theconcept of using glutamine synthetase to detoxify ammonia by convertingit to the non-toxic product glutamine. In particular, the presentinvention proposes systemically to administer glutamine synthetase as aprotein therapy to reduce the levels of ammonia in the blood.

Glutamine synthetase (GS) catalyses the reaction:

Glutamate+ATP+NH₃→Glutamine+ADP+phosphate.

Glutamine synthesis occurs in a number of organs of the body and mayplay a role in organ and whole-body nitrogen balance. Very recently,gene therapy based on overexpression of GS in the skeletal muscle hasbeen proposed for the treatment of acute hyperammonemia (Torres-Vega etal, Gene Therapy 2015, 22, 58-64), the rationale for this therapy beingto replace or augment GS which is generally deficient in the muscles ofpatients with liver disease, thereby aiming to increase the clearance ofammonia by this enzyme in the muscle. Gene therapy has however proveddifficult to successfully administer in clinical practice and not allpatients are suitable for it (e.g. children, with the exception ofstem-cell based gene therapies), or patients may be refractory to genetherapy (e.g. for immune reasons). Further, such a therapy would producea mainly localised effect, in the muscle. Accordingly, a need stillremains for a more generally applicable therapy.

Administration of GS systemically as a protein may serve to achieve sucha more general effect. We have shown that GS, particularly human GS, maysuccessfully be expressed and purified and may retain, or demonstrate,GS activity, both in unmodified and modified form, conjugated to apolymeric partner such as polyethylene glycol. Further, animal studieshave shown that high circulating levels of the GS may be achieved bysystemic administration and that both modified and unmodified GSadministered to the animal retains activity in the blood and othertissues (e.g. liver). Thus, therapeutic levels of GS may be achieved bysystemic (e.g. parenteral) administration of GS protein.

Additionally, the inventors have also surprisingly found that thecombined use of a GS protein and an ammonia lowering agent (such as anitrogen scavenger, for example, a pharmaceutically acceptable salt ofphenylacetic acid, such as sodium phenylacetate) has a synergisticeffect and may further increase the ability of the GS protein to treator prevent hyperammonemia. Accordingly, in one aspect the presentinvention provides a glutamine synthetase (GS) protein for use intreating or preventing hyperammonemia by systemic non-oraladministration to a subject.

In a suitable embodiment, the GS protein for use in treating ofpreventing hyperammonemia, may be for use in combination with an ammonialowering agent.

In another aspect, the invention provides an ammonia lowering agent foruse in combination with a GS protein, for use in treating or preventinghyperammonemia.

A related aspect of the invention also provides use of a GS protein forthe manufacture of a composition (e.g. a pharmaceutical or nutritionalcomposition, for example a medicament or supplement) for the treatmentor prevention of hyperammonemia by systemic non-oral administration to asubject.

In a suitable embodiment, the composition may be for use in combinationwith an ammonia lowering agent.

A further aspect of the invention also provides use of an ammonialowering agent for the manufacture of a composition for use incombination with a GS protein for the treatment or prevention ofhyperammonemia.

In a further aspect the present invention provides use of a GS proteinand an ammonia lowering agent for the manufacture of a composition foruse in the treatment or prevention of hyperammonemia.

In a further aspect the present invention provides a method of treatingor preventing hyperammonemia in a subject, said method comprisingsystemically and non-orally administering a GS protein to a said subject(more particularly to a subject in need thereof). In a suitableembodiment, the method further comprises administering an ammonialowering agent.

Also provided is a composition comprising a GS protein, for use intreating or preventing hyperammonemia by systemic non-oraladministration to a subject.

Suitably, the composition comprising a GS protein may be apharmaceutical or nutritional composition, for example a medicament orsupplement. Suitably, the composition may further comprise an ammonialowering agent.

The invention also relates to a composition comprising an ammonialowering agent, for use in treating or preventing hyperammonemia.Suitably, the composition comprising an ammonia lowering agent may be apharmaceutical or nutritional composition, for example a medicament orsupplement.

The invention also relates to a composition comprising a GS protein andan ammonia lowering agent.

Suitably the composition may be a pharmaceutical or nutritionalcomposition. Suitably, the composition may be for use in treating orpreventing hyperammonemia.

The term “GS protein” may alternatively be expressed as “a proteinhaving glutamine synthetase (GS) activity”. The term “protein” is usedbroadly used herein to include any proteinaceous molecule, includingpeptides and polypeptides, as well as protein or polypeptide fragments;the GS protein does not have to be, or to correspond to, a full lengthGS enzyme as it appears in nature (e.g. a native or wild-type GS) andtruncated or other variants are included, as described more detailbelow. Also included are conjugates, or fusions, of the GS protein withother molecules, as also described in more detail below.

The term “hyperammonemia” includes any condition in which ammonia in theblood (or as measured or determined in any blood-derived product orsample e.g. blood plasma) is elevated as compared to the level ofammonia in a subject without the condition, e.g. a healthy subject or asubject without the underlying condition which leads to or causes thehyperammonemia. In health, ammonia transport and metabolism are tightlyregulated to maintain a low plasma/blood concentration (normal range10-40 μmol/L). Thus, a plasma (or blood) ammonia concentration of >40,60 or 70 or 80 μmol/L or more, e.g. 41, 42, 45, 50, 55, 60, 70 or 80μmol/L or more may be viewed as indicative of hyperammonemia. Forexample, a plasma ammonia concentration of >100 μmol/L, and particularly150 μmol/L or higher, where associated with a normal anion gap and anormal plasma glucose concentration, may be indicative ofhyperammonemia, or more particularly, indicate the presence of a UCD.

Hyperammonemia may arise from any of the causes or conditions discussedabove, i.e. it may be congenital or acquired, primary or secondary, asdiscussed above. Thus, in one embodiment the hyperammonemia may arisedue to (or be associated with) a urea cycle disorder (UCD). As discussedabove, the UCD may arise from a defect in any one or more of theproteins of the urea cycle, which may inactivate or reduce the activityof the protein.

In a further embodiment the hyperammonemia may arise due to an inbornerror of metabolism affecting a protein (e.g. enzyme) which is not partof the urea cycle, but which affects nitrogen metabolism and/or balancein the body, and leads to an increase in the amount of ammonia in theblood. Suitably, such an inborn error of metabolism may be glutaminesynthetase deficiency.

In still further embodiments, the hyperammonemia may be acquired and mayarise to disease or damage to an organ or tissue of the body which isinvolved in nitrogen metabolism and/or balance, for example incatabolism and/or excretion of nitrogen-containing molecules orsubstances e.g. the liver or kidney.

Thus any kind of liver damage or disease, including both chronic oracute liver failure, for example liver damage due to excess alcoholconsumption or due to drugs (whether recreational or medicinal),cirrhosis of the liver due to any cause, non-alcoholic fatty liverdisease, infection of the liver or trauma to the liver may lead tohyperammonemia.

Similarly, any kind of damage to or disease of the kidney is describedfor the liver above may also lead to hyperammonemia. Thus, any conditionaffecting multiple organs of the body (e.g. multiple organ failure) suchas for example sepsis, organ damage from injury (whether external injurye.g. trauma, or internal injury e.g. from autoimmune disorders), or anysystemic infection, may give rise to hyperammonemia.

Hyperammonemia may be detected or diagnosed based on clinical,biochemical and/or molecular genetic data, depending upon the underlyingcause. Thus for example it may be detected by assessing or monitoringblood levels of ammonia (e.g. in plasma or serum or any blood-derivedsample) according to techniques well known and used in the art. Analysisof amino acids present in the blood (plasma or serum, etc.) and/or theconcentration thereof (for example arginine or citrulline) or othermetabolites in the blood or other body fluids or tissues (e.g. oroticacid in urine) may also help to identify that a UCD is involved, and/orwill determine the precise nature thereof (i.e. the specificprotein/enzyme defect involved). Such determinations and analyses may becombined with clinical assessments, e.g. neurological andneuropsychiatric evaluations, including both physical (e.g. MRI or otherimaging) and/or behavioural/response tests etc., liver and/or kidney orother organ function tests etc. In the case of a suspected UCD,investigations of family history and/or molecular genetic tests and/orassessments of enzyme activity of the urea cycle enzymes may also beundertaken.

As used herein, reference to a glutamine synthetase or GS protein foruse according to the present invention includes reference to all formsof enzymatically-active GS, including human GS and GS from non-humananimals (such as mouse, cow, rabbit, rat, monkey, chimpanzee, and dogetc), or from other sources including for example fungi, plants orbacteria, as well as enzymatically-active variants. Representative GSproteins thus include those having at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity with the GS polypeptide set forth in SEQ ID NO:1 or 2or 4 (human GS, precursor, mature and N-terminal tagged formsrespectively) or with the GS polypeptide set forth in SEQ ID NO. 6 (GSfrom Lactobacillus acididophilus strain 30SC) or SEQ ID NO. 7 (GS fromcorn, Zea mays), or an enzymatically-active fragment thereof. Forexample, reference to GS may also include N- and/or C-terminallytruncated polypeptides or amino acid modified protein (eg.post-translational modifications, such as adenylation, or othermodifications such as amino acid polymorphisms that may affect thestructure or activity of the protein). It may also include multimers ofthe protein. The term “GS” thus includes all native forms of GS enzymesor polypeptides as well as enzymatically-active fragments or variantsthereof, including synthetically derived and modified polypeptideshaving one or more amino acid substitutions, additions (includinginsertions and extensions) or deletions, which retain GS enzymaticactivity.

The GS may thus be, or may be derived from, any enzyme falling withinthe enzyme classification EC 6.3.1.2. It may be any polypeptide orpeptide having GS activity. GS activity may be defined as the ability toconvert glutamate and ammonia to glutamine, e.g. according to thereaction scheme set out above. GS activity may be assessed or determinedusing assays or tests (e.g. a functional activity assay) as known in theart and described in the literature. For example, GS enzyme activityassays are described in Listrom et al, Biochem. J. 1997, 328, 159-163.An assay for GS activity is also described in the Examples below (seeExamples 2 and 4).

The term also includes a pro-drug for the GS, that is a form which doesnot in itself exhibit GS activity, but which may be converted to anactive GS upon administration to the subject.

Thus, as used herein, “enzymatically-active” with reference to a GSprotein or polypeptide refers to a GS protein or polypeptide that cancatalyze the conversion of glutamate and ammonia to glutamine.Typically, the enzymatically-active GS protein or polypeptide exhibitsat least or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%or 99% of the enzymatic activity of a GS polypeptide as set forth in SEQID NO:1, 2, 4, 6 or 7.

The term “subject” as used herein includes any human or non-human animaland particularly refers to mammals, including for example humans,primates, livestock animals (e.g. sheep, pigs, cattle, horses, donkeys),laboratory test animals (eg. mice, rabbits, rats, guinea pigs),companion animals (eg. dogs, cats) and captive wild animals (eg. foxes,kangaroos, deer). Preferably, the mammal is human or a laboratory testanimal. Even more preferably, the mammal is a human.

As used herein the terms “treating”, “treatment”, “preventing” and“prevention” refer to any and all uses which remedy or ameliorate acondition or symptoms, prevent the establishment of a condition ordisease, or otherwise prevent, hinder, retard, reduce or reverse theprogression of a condition or disease or other undesirable symptoms inany way whatsoever. Thus the terms “treating” and “preventing” and thelike are to be considered in their broadest context. For example,treatment does not necessarily imply that a patient is treated untiltotal recovery, but includes any improvement or amelioration in thecondition of a patient or subject, or in a symptom of the disease orcondition. Thus for example in the case of a UCD, treatment according tothe present invention does not of course treat the underlying geneticdisorder, but rather the resulting clinical condition of hyperammonemia.In conditions which display or are characterized by multiple symptoms,the treatment or prevention need not necessarily remedy, ameliorate,prevent, hinder, retard, reduce or reverse all of said symptoms, but mayremedy, ameliorate, prevent, hinder, retard, reduce or reverse one ormore of said symptoms. In a suitable embodiment, “treatment” accordingto the present invention a reduction in the levels of ammonia in theblood, to for example normal or healthy levels, e.g. to the range 10-40μmol/L. Thus, treatment includes the restoration of normal or healthyammonia levels. Similarly, prevention according to the present inventionmay include maintenance of plasma/blood ammonia levels in any normal orhealthy range, as indicated above.

In a suitable embodiment, “treatment” according to the present inventionmay include an increase in glutamine synthetase levels and/or activityin subject's tissue, to for example normal or healthy levels. Such anincrease may be in any suitable tissue (for example liver and/ormuscle). An increase in glutamine synthetase levels and/or activity maybe, for example, determined by measuring levels of glutamine, levels ofglutamate, and/or determining the ration between levels of glutamine andlevels of glutamate, in a subject. It will be appreciated that normal orhealthy levels of glutamine and/or glutamate may vary depending on thesample in which these are measured. It will also be appreciated thatnormal or healthy levels of glutamine and/or glutamate may be subjectspecific, and depend on factors such as the subject's weight, diet, sexand age. Normal or healthy levels of glutamine and/or glutamate will beknown to those skilled in the art.

In a suitable embodiment “treatment” according to the present inventionmay include a reduction in oedema. The term “oedema” as used hereinrefers to abnormal accumulation of serous fluid in the subject. In asuitable embodiment, oedema may be brain oedema, pulmonary oedema,peripheral oedema, and/or macular oedema. Suitably, in the context ofthe present invention, “treatment” may refer to a reduction in brainoedema, for example in the prefrontal cortex. By way of example, oedemamay be assessed by a CT scan, MRI, and/or an x-ray. Other methods ofassessing oedema will be known to those skilled in the art.

In a suitable embodiment “treatment” according to the present inventionmay include an improvement in neuropsychological, neuropsychiatric andneurocognitive function. The term “neuropsychological, neuropsychiatricand neurocognitive function” refers to the function of the brain whichcontrols, for example, memory, attention, cognition, psychomotoractivity, coordination and mood. Uses, methods and compositions of theinvention which involve the combination of a GS protein and an ammonialowering agent may be particularly useful in such an embodiment. Variousmethods for assessing neural function will be known to those skilled inthe art. The neuronal functional status may be assessed usingelectroencephalography, computerised tests, paper and pencil tests andassessments by a neuropsychologist.

In a suitable embodiment “treatment” according to the present inventionmay include an improvement in sarcopenia and physical function. The term“sarcopenia” refers to reduced muscle mass. The term “physical function”refers to the subject's strength, in particular muscle strength. Theseverity of sarcopenia may be determined by using clinical tools,nutritional tools, measurement of body composition or imaging. Physicalfunction may be assessed, for example, by assessment of oxygen deliveryand consumption, exercise test and hand-grip test, and/or an improvementin levels of fatigue, and/or immune system function.

As used herein, “amelioration” refers to the lessening of severity of atleast one indicator or symptom of a condition or disease. In certainembodiments, amelioration includes a delay or slowing in the progressionof one or more indicators of a condition or disease. The severity ofindicators may be determined by subjective or objective measures, whichare known to those skilled in the art.

As used herein the term “associated with” when used in the context of adisease or condition “associated with” the elevated levels of ammoniameans that the disease or condition may result from, result in, becharacterised by, or otherwise associated with the elevated levels ofammonia. Thus, the association between the disease or condition and theelevated levels of ammonia may be direct or indirect and may betemporally separated.

Appropriate samples for determination of levels of ammonia include anyappropriate or desired sample in which the ammonia may occur. By thesame token, appropriate samples for determination of glutaminesynthetase levels and/or glutamine synthetase activity, include anyappropriate or desired sample in which glutamine synthetase, glutamineand/or glutamate may be present. These may be any appropriate or desiredtissue or body fluid sample. An example of a suitable tissue is liverand/or muscle tissue. Conveniently, a sample may be any body fluidsample, and typically will be blood or any blood-derived sample e.g.plasma or serum etc, but it may be any other body fluid e.g. urine,cerebrospinal fluid, or a stool or tissue sample etc, for example abiopsy sample or a lavage or washing fluid sample etc. This may dependof course on the precise nature of the condition to be treated etc.

As used herein the term “effective amount” includes within its meaning anon-toxic but sufficient amount or dose of, depending on the context, GSprotein and/or an ammonia lowering agent, to provide the desired effect.It will be appreciated that the effective amount of the protein and/orthe ammonia lowering agent may be different. Exemplary therapeuticallyeffective amounts are specified elsewhere in this specification.

The exact amount or dose required will vary from subject to subjectdepending on factors such as the species being treated, the age andgeneral condition of the subject, the severity of the condition beingtreated, the particular FMO3 being administered and the mode ofadministration and so forth. Thus, it is not appropriate to specify anexact “effective amount”. However, for any given case, an appropriate“effective amount” may be determined by one of ordinary skill in the artusing only routine experimentation.

Human GS is expressed as a polypeptide of 373 amino acids (as shown inSEQ ID NO. 1). This represents the full “precursor” protein as expressedwhich is then further processed to a mature form having amino acids2-373 (only the N-terminal methionine is removed to yield a matureprotein of 372 amino acids in vivo, as shown in SEQ ID NO. 2. Theexemplary polynucleotide set forth in SEQ ID NO. 3 represents a cDNAencoding the polypeptide of SEQ ID NO. 1. SEQ ID NO. 4 represents amodified human GS protein, comprising the GS polypeptide of SEQ ID NO. 1provided with an N-terminal His-tag and linker sequence, as prepared andused in the Examples below. SEQ ID NO. 5 is a cDNA sequence encoding thepolypeptide of SEQ ID NO. 4, codon-optimised for expression in bacteria,as used in the Examples below.

Human GS has been well-characterised (see for example Listrom et al.,1997, supra). GS enzymes from other organisms, including plants andbacteria, have also been identified, and nucleic acid and amino acidsequences of such other GS enzymes are well known in the art andprovided in freely-available databases, such as, for example, theNational Center for Biotechnology Information (NCBI) Nucleotide(ncbi.nlm.nih.gov/nuccore) and Protein (ncbi.nlm.nih.gov/protein)databases. Although the sequence identity between plant or bacterial GSenzymes and human GS may be low, the structural and functionalsimilarity is high.

Accordingly, plant or bacterial GS, or indeed GS from other organisms,or amino acid sequence variants thereof, may be used. By way ofrepresentative example, SEQ ID NO. 6 sets forth the amino acid sequenceof the GS from Lactobacillus acidophilus strain 30SC, which has 23.8%sequence identity with human GS and 61.9% sequence identity with the GSof Lactobacillus casei, and SEQ ID NO. 7 sets forth the amino acidsequence of the GS from corn (maize, Zea mays), which has 55.7% sequenceidentity with human GS.

GS commonly occurs as a multimer comprising multiple (i.e. 2 or more)monomer subunits. The GS amino acid sequence provided above, forexample, represents such a monomer subunit. Human GS is most frequentlyreported as a dodecamer (12 subunits). As used herein, the GS may beprovided as a monomer and/or as a multimer. The multimer may comprise 2or more monomer subunits, for example 2 to 20, 2 to 16, 2 to 15, 2 to 14or 2 to 12 subunits.

Indeed, a surprising feature of the present invention is that contraryto the reports in the literature of a 12-subunit multimer, human GS maybe expressed and/or obtained as a mixture of multiple different types ofmultimer, including also a monomeric form. The monomeric form has beenshown to be active. Thus, according to the present invention, the GS maybe used as a monomer and/or as a multimer, and the multimer may beprovided as a single multimeric form, or as a mixture of differentmultimeric forms which may or may not include the monomer. As reportedin the examples below, 4 or more, e.g. 4 to 10, e.g. 5 to 8 multimericforms may be obtained. The multimers may range in size from 2 to 20subunits.

The GS protein used in the methods provided herein can be obtained byany method known in art, such as recombinant methods, protein isolationand purification methods and chemical synthesis methods, providing theresulting GS exhibits enzymatic activity. Accordingly, the GS can berecombinant GS, native GS isolated from tissue, or chemicallysynthesised GS.

It is well within the capabilities of a skilled person to modify a GSpolypeptide, such as the polypeptide set forth in SEQ ID NO:1, togenerate enzymatically-active GS variants for use in the methodsprovided herein. For example, a person skilled in the art wouldunderstand that modifications at positions involved in substratebinding, or in the active site, are less likely to be tolerated thanmodifications at positions outside these critical regions. Any GSpolypeptide can be tested using methods well known in the art, such asthose described in the Examples below, to assess the ability of the GSpolypeptide to catalyze the conversion of glutamate and ammonia toglutamine.

In some examples, the GS used according to the invention herein isrecombinant GS produced using prokaryotic or eukaryotic expressionsystems well known in the art. Exemplary prokaryotic expression systemsinclude, but are not limited to, Escherichia coli expression systems,and exemplary eukaryotic expression systems include, but are not limitedto, yeast, insect cell and mammalian cell expression systems.

Nucleic acid encoding GS can be obtained by any suitable method,including, but not limited to, RT-PCR of liver RNA and syntheticnucleotide synthesis. Primers for amplification can be designed based onknown GS sequences, such as that set forth above. Nucleic acid and aminoacid sequences of GS are well known in the art and provided infreely-available databases, such as, for example, the National Centerfor Biotechnology Information (NCBI) Nucleotide(ncbi.nlm.nih.gov/nuccore) and Protein (ncbi.nlm.nih.gov/protein)databases.

Nucleic acid encoding the GS polypeptide, such as nucleic acid having asequence set forth in SEQ ID NO. 3, can be cloned into an expressionvector suitable for the expression system of choice. In some instances,the nucleic acid is codon-optimized for expression in a particularsystem. For example, the nucleic acid encoding the GS polypeptide can becodon-optimised for expression in E. coli. An exemplary codon-optimisednucleic acid encoding GS for expression in E. coli is set forth in SEQID NO 5, which encodes a GS polypeptide comprising a His-tag attachedvia a GGGGS linker (as set forth in SEQ ID NO. 4).

Typically the nucleic acid encoding the GS is cloned into an expressionvector, operably linked to regulatory sequences that facilitateexpression of the heterologous nucleic acid molecule. Many expressionvectors suitable for the expression of GS are available and known tothose of skill in the art. The choice of expression vector is influencedby the choice of host expression system. Such selection is well withinthe level of skill of the skilled artisan. In general, expressionvectors can include transcriptional promoters and optionally enhancers,translational signals, and transcriptional and translational terminationsignals. Expression vectors that are used for stable transformationtypically have a selectable marker which allows selection andmaintenance of the transformed cells. In some cases, an origin ofreplication can be used to amplify the copy number of the vectors in thecells.

GS polypeptides also can be expressed as protein fusions. For example, afusion can be generated to add additional functionality to apolypeptide. Examples of fusion proteins include, but are not limitedto, fusions containing GS and an affinity tag for purification (e.g. ahis-tag e.g. his6, MYC, FLAG, HA or GST tag), a leader sequence (such asthe pelB leader sequence), a sequence for directing protein secretion,or a protein for stabilising and/or solubilising GS (e.g.maltose-binding protein (MBP)), or a protein for increasing in vivohalf-life (e.g. albumin or Fc domains, or fragments thereof).

Prokaryotes, especially E. coli, provide a system for producing largeamounts of GS. Transformation of E. coli is a simple and rapid techniquewell known to those of skill in the art. Expression vectors for E. colican contain inducible promoters that are useful for inducing high levelsof protein expression and for expressing proteins that exhibit sometoxicity to the host cells. Examples of inducible promoters include thelac promoter, the trp promoter, the hybrid tac promoter, the T7 and SP6RNA promoters and the temperature regulated APL promoter.

In other examples, eukaryotic expression systems are used to produce theGS, such as baculovirus expression systems. Typically, expressionvectors use a promoter such as the polyhedrin promoter of baculovirusfor high level expression. Commonly used baculovirus systems includebaculoviruses such as Autographa californica nuclear polyhedrosis virus(AcNPV), and the Bombyx mori nuclear polyhedrosis virus (BmNPV) and aninsect cell line such as Sf9 derived from Spodoptera frugiperda,Pseudaletia unipuncta (A7S) and Danaus plexippus (DpNI). For high levelexpression, the nucleotide sequence of the GS is fused immediatelydownstream of the polyhedrin initiation codon of the virus.

Yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe,Yarrowia lipolytica, Kluyveromyces lactis, and Pichia pastoris can alsobe used expression hosts for GS. Yeast can be transformed with episomalreplicating vectors or by stable chromosomal integration by homologousrecombination. Typically, inducible promoters, such as include GALI,GAL7, and GALS, are used to regulate gene expression. Yeast expressionvectors often include a selectable marker such as LEU2, TRPI, HIS3, andURA3 for selection and maintenance of the transformed DNA.

Mammalian expression systems also can be used to express GS. Expressionconstructs can be transferred to mammalian cells by viral infection suchas adenovirus or by direct DNA transfer such as liposomes, calciumphosphate, DEAE-dextran and by physical means such as electroporationand microinjection. Expression vectors for mammalian cells typicallyinclude an mRNA cap site, a TATA box, a translational initiationsequence (Kozak consensus sequence) and polyadenylation elements. Suchvectors often include transcriptional promoter-enhancers for high levelexpression, for example the SV40 promoter-enhancer, the humancytomegalovirus (CMV) promoter, and the long terminal repeat of Roussarcoma virus (RSV). Exemplary cell lines available for mammalianexpression include, but are not limited to, mouse, rat, human, monkey,and chicken and hamster cells, such as BHK, 293-F, CHO, Balb/3T3, HeLa,MT2, mouse NSO (non-secreting) and other myeloma cell lines, hybridomaand heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS,NIH3T3, HEK293, 293S, 293T, 2B8, and HKB cells.

Following expression, GS can be purified using any method known to thoseof skill in the art including, but not limited to, SDS-PAGE, sizefraction and size exclusion chromatography, ammonium sulfateprecipitation, chelate chromatography, ionic exchange chromatography andaffinity chromatography. Affinity purification techniques can be used toimprove the efficiency and purity of the preparations. For example,antibodies and other molecules that bind GS can be used in affinitypurification. As discussed above, expression constructs can beengineered to add an affinity tag such as a his, myc, FLAG or HA tag orGST moiety to GS, which can then be affinity purified with Ni-resin, mycantibody, HA antibody, FLAG antibody or glutathione resin, respectively.Purity can be assessed by any method known in the art including gelelectrophoresis and staining and spectrophotometric techniques, such asSDS page and Size Exclusion Chromatography (SEC).

For use according to the present invention, the affinity tag (e.g. histag etc) may be removed, but this is not necessary, and the GSpolypeptide may be used with the tag attached.

The tag or other fusion partner may be attached to the GS via a linker,which may be any suitable linker, according to principles well known inthe art. Such a linker may typically and conveniently be a short (e.g. 2to 10, 2 to 8 or 2-6 mer) peptide. By way of example the linker GGSG maybe mentioned, but could be made up of any suitable amino acids. Aminoacid linkers enable preparation of the fusion protein by recombinantmeans but non-amino acid-based linkers might also be used, againaccording to principles and techniques well known in the art anddescribed in the literature. The linker may be cleavable (e.g.enzymatically) or non-cleavable.

GS polypeptides can be prepared as naked polypeptide chains or asmodified polypeptides which are modified by coupling or conjugating to afurther moiety or chemical group or substance. Exemplary modificationsinclude, but are not limited to, pegylation, albumination or other knownmodifications. For example, in some instances, the GS polypeptides foruse in the described methods are peglyated using standard methods wellknown in the art. This may serve, for example, to improve the half-lifeof the GS protein in the circulation. Thus, in a preferred embodiment ofthe invention the GS protein may be provided as a conjugate with apolymer such as polyethylene glycol (PEG) or a poly- or oligosaccharide.Conjugates with PEG are particularly preferred. As indicated above thepreparation of such conjugates is well known in the art and described inthe literature. Thus, PEGs of various sizes may be used to prepare theconjugates e.g. ranging from 100 Daltons to 100 kD, but more often from5 kD to 100 kD, for example 12 or 15 kD to 60 or 80 kD, such as 15 to50, 15 to 40, or 15 to 30 kD. Further, the PEG may be attached or linkedto the GS protein in various ways, and more than one PEG may be attachedto each single protein. It may be linked directly or indirectly, e.g.via a linker as described, for fusion proteins above or by any molecularor chemical group which may provide a linker function. Thus, the PEG maybe linked at one or both of the N- or C-termini, or internally in the GSmolecule, for example at the amino group of one or more lysine residuesin the GS protein molecule or at any other chemical moiety or residue inthe protein molecule. Methods for coupling or conjugating polymers suchas PEG to proteins are well known in the art and described in theliterature (see for example Roberts et al. 2012, Advanced Drug DeliveryReviews, 64 (supplement) 116-127 and Veronese 2001, Biomaterials 22,405-417). The data presented in the Examples below shows that PEGconjugates prepared by linking the PEG to the N-terminal are especiallyeffective, for example in activity assays of liver lysates from animalsadministered various conjugates. Accordingly, a PEG conjugate comprisinga PEG linked to the N terminus of a GS protein represents one preferredembodiment of the present invention. The GS protein may be pegylated inmonomeric and/or multimeric form. Thus, for convenience a preparationcomprising both monomeric and various multimeric forms of GS may besubjected to pegylation.

GS can be formulated as a pharmaceutical composition for administrationto a subject. GS can be formulated in any conventional manner by mixinga selected amount of GS with one or more physiologically orpharmaceutically acceptable carriers or excipients.

Accordingly, a further aspect of the invention provides a pharmaceuticalcomposition comprising a GS protein and one or morepharmaceutically-acceptable carriers or excipients, wherein thecomposition is for non-oral systemic administration.

Selection of the carrier or excipient is within the skill of theadministering profession and can depend upon a number of parameters,such as the mode of administration. In some examples the GS is providedas a fluid. In other instances, the GS is provided in dried form, suchas desiccated or freeze-dried form. Such dried forms can be rehydratedprior to administration by the addition of a suitable solution, such aswater, buffer, saline or other suitable solution. The GS provided hereincan be formulated for direct administration or can be formulated fordilution or other modification. Accordingly, the GS can be formulated insingle (or unit) dosage forms or multiple dosage forms. Examples ofsingle dose forms include ampoules and syringes. Examples of multipledose forms include vials and bottles that contain multiple unit doses.

The concentrations of the GS in the formulations are effective fordelivery of an amount of GS that, upon administration, is effective toconvert ammonia to glutamine in the presence of glutamate. Theconcentrations and amounts will depend on several factors, including thelevels of the substrates in the subject, and mode of administration, andcan be empirically determined. Exemplary concentrations of GS in thecompositions provided herein include, but are not limited to,concentrations of or about 0.1, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 2000,3000, 4000 or 5000 mg/mL GS or more.

To formulate the GS composition, in one embodiment the weight fractionof GS is dissolved, suspended, dispersed, or otherwise mixed in aselected vehicle at the desired concentration. The resulting mixturesare solutions, suspensions, emulsions and other such mixtures, and canbe formulated as a non-aqueous or aqueous mixture, including but notlimited to, a solution, suspension, paste, gel, aerosol, spray, or anyother formulation suitable for systemic administration.

Generally, the GS composition is prepared in view of approval from aregulatory agency or otherwise prepared in accordance with generallyrecognized pharmacopeia for use in animals and in humans. The GScomposition can include carriers such as a diluent, excipient, orvehicle. Such pharmaceutical carriers can be sterile liquids, such aswater and oils. Saline solutions and aqueous dextrose and glycerolsolutions also can be employed as liquid carriers, particularly forinjectable solutions. Compositions can contain along with an activeingredient: a diluent such as lactose, sucrose, dicalcium phosphate, orcarboxymethylcellulose; a lubricant, such as magnesium stearate, calciumstearate and talc; and a binder such as starch, natural gums, such asgum acaciagelatin, glucose, molasses, polvinylpyrrolidine, cellulosesand derivatives thereof, povidone, crospovidones and other such bindersknown to those of skill in the art. Suitable pharmaceutical excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, andethanol. A GS composition, if desired, also can contain minor amounts ofwetting or emulsifying agents, or pH buffering agents, for example,acetate, sodium citrate, cyclodextrine derivatives, sorbitanmonolaurate, triethanolamine sodium acetate, triethanolamine oleate, andother such agents. Examples of suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin.

Liposomal suspensions, including tissue-targeted liposomes, can also besuitable as pharmaceutically acceptable carriers. These can be preparedaccording to methods known to those skilled in the art. Liposomaldelivery can also include slow release formulations, includingpharmaceutical matrices such as collagen gels and liposomes modifiedwith fibronectin.

As well as in pharmaceutical compositions, the GS may also be formulatedor administered in other ways, for example in a nutritional compositionsuch as a dietary supplement, for example a parenteral nutritioncomposition (e.g. alone or alongside other supplement ingredients). Itmay be included in such foods as a polypeptide (e.g. purified enzyme) oras part of an expressing host cell or organism. Thus for examplemicrobial (e.g. yeast or bacterial or fungal) host cells or plants(including plants cells) may be engineered to express GS and may beadministered as such, e.g. a whole cells or extracts or other processedproducts (in which enzymatic activity may be retained), or may beincorporated into nutritional compositions. Thus, for example, bacterialor yeast cells suitable for human or non-human animal consumption may beengineered to express GS (namely by introduction of a nucleic acidmolecule comprising a nucleotide sequence encoding GS). Alternatively,plants may be engineered in an analagous manner and appropriate plantparts etc (e.g. seeds, leaves, tubers etc) may be provided foradministration. It is known in the art which microoganisms (yeasts,bacteria, algae or fungi for example) are suitable for human or otheranimal consumption and many such organisms are used today, for examplein probiotic formulations. Any such probiotic organisms or formulationscould be used e.g. based on lactic acid bacteria such as Bifidobacteriumor Lactobacillus sp. (e.g. L. acidophilus) etc. Thus, according to thepresent invention such organisms or preparations may be formulated forand administered directly into the GI tract, e.g. by injection orinfusion, or enema or rectal administration etc. The precise amount ordose of the GS administered to the subject depends on the activity ofthe GS, the route of administration, the disease or condition beingtreated, the number of dosages administered, and other considerations,such as the weight, age and general state of the subject. Particulardosages and administration protocols can be empirically determined orextrapolated from, for example, studies in animal models. Exemplarytherapeutically effective doses of the GS include, but are not limitedto, from or from about 0.1 μg/kg body weight per day to or to about10000 μg/kg body weight per day, including from or from about 1 μg/kg toor to about 1000 μg/kg body weight per day, or from or from about 10μg/kg to or to about 100 μg/kg body weight per day Thus, for example, asubject can be administered 0.1, 0.2, 0.3, 0.4, 0.5, 1, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 400, 600, 800, 1000, 2000, 4000, 6000,8000, 10000, or 20000 μg or more the GS per kg body weight per day.

It is a feature of the present invention that the GS is administeredsystemically, but non-orally. By “orally” it is meant per-oral delivery.Accordingly, non-oral means that the GS protein is not administered byingestion via the mouth. Although other means of administration whichdeliver the GS protein to the intestines or more generally thegastrointestinal (GI) tract may be included (e.g rectally, or by enema,or direct administration into the GI tract), in certain embodiments theyare excluded. Accordingly, in a certain embodiment the inventionincludes enteral administration, but in another embodiment it does not.In a further embodiment the invention does not include administration tothe muscle, particularly to skeletal muscle. Thus, in such an embodimentthe administration is not directed to muscle, i.e. is a non-muscledirected therapy.

Accordingly the GS can be administered by any method and route thatdelivers the GS protein systemically to the body, but does not involveoral administration. In certain embodiments the GS protein may beadministered parenterally. A skilled artisan would readily understandand be able to select appropriate modes of administration or delivery,including, but not limited to, intravenous, intramuscular, intradermal,transdermal, subcutaneous, or intraperitoneal administration, as well asby any combination of any two or more thereof, formulated in a mannersuitable for each route of administration. In some instances, the GScompositions described herein are administered subcutaneously. In otherinstances the GS compositions may be administered intravenously. Forexample, the GS compositions can be administered intravenously byinjection or infusion, such as by an intravenous bolus.

The GS protein may also be administered in conjunction or combinationwith other therapeutic or active agents, notably a second or furthertherapeutic agent which may treat (e.g. to improve) hyperammonemia. Asecond or further therapeutic agent which is active to treathyperammonemia may be alternatively defined as an anti-hyperammoneniaagent or as an agent against hyperammonemia. The second or further agentmay typically be an ammonia lowering agent such as a nitrogen scavenger(or an ammonia scavenger) or a replacement amino acid or urea cycleintermediate, or an analogue thereof. Such an agent may thereforeinclude an amino acid, for example arginine, glutamate, citrullineand/or ornithine, and/or N-acetyl glutamate and/or the analogue moleculecarbamyl glutamate (Carbaglu®). More suitably, the second or furtheragent is an ammonia lowering agent, more suitably a nitrogen scavenger.Such an embodiment gives rise to certain aspects of the presentinvention.

The term “ammonia lowering agent” refers to a compound which removesammonia and/or reduces, or inhibits ammonia production. An ammonialowering agent may be selected from the group consisting of a nitrogenscavenger, an ion exchange resin (for example Relapsa), an ammoniaabsorber (such a liposomal based ammonia absorber, for exampleVersantis), an engineered microbiome that removes ammonia (for exampleSynlogic), Rifaximin and Lactulose.

The term “nitrogen scavenger” as used herein, refers to a compound whichreduces the levels of nitrogen and/or ammonia in a subject by removingammonia. In a suitable embodiment the nitrogen scavenger reduces theamount of nitrogen and/or ammonia in the subject by being metabolised tophenylacetyl glutamine, which may be then excreted in urine.

In a suitable embodiment, a nitrogen scavenger may be selected from thegroup consisting of a pharmaceutically acceptable salt of phenylaceticacid (also referred to herein as phenylacetate), a pharmaceuticallyacceptable salt of phenylbutyric acid (also referred to herein asphenylbutyrate), glycerol phenylbutyrate, a pharmaceutically acceptablesalt of benzoic acid, a pharmaceutically acceptable pro-drug thereof,and ammonia binding resin. Other nitrogen scavengers will be well knownto those skilled in the art.

As used herein, the term “pharmaceutically-acceptable salt” includes,for example, an acid-addition salt of phenylacetic acid which issufficiently basic, for example, an acid-addition salt with, forexample, an inorganic or organic acid, for example hydrochloric,hydrobromic, sulfuric, phosphoric, trifluoroacetic, formic, citricmethane sulfonate or maleic acid. In addition, a suitablepharmaceutically acceptable salt of phenylacetic acid which issufficiently acidic is an alkali metal salt, for example a sodium orpotassium salt, an alkaline earth metal salt, for example a calcium ormagnesium salt, an ammonium salt or a salt with an organic base whichaffords a pharmaceutically acceptable cation, for example a salt withmethylamine, dimethylamine, trimethylamine, piperidine, morpholine ortris-(2-hydroxyethyl)amine.

In a suitable embodiment, a pharmaceutically acceptable salt ofphenylacetic acid may be selected from the group consisting of sodiumphenylacetate, potassium phenylacetate, ornithine phenylacetate.

In a suitable embodiment, a pharmaceutically acceptable salt ofphenylbutyric acid may be selected from the group consisting of sodiumphenylbutyrate and potassium phenylacetate.

In a suitable embodiment, a pharmaceutically acceptable salt of benzoicacid may be selected from the group consisting of sodium benzoate andpotassium benzoate.

It shall be understood that an ammonia lowering agent may exist insolvated as well as unsolvated forms such as, for example, hydratedforms. It is to be understood that in the context of the presentinvention encompassed are all such solvated and unsolvated forms.

The term “pro-drug” as used herein refers to an agent rendered lessactive by a chemical or biological moiety than the ammonia loweringagent (such as nitrogen scavenger), but which metabolises into orundergoes in vivo hydrolysis to form the ammonia lowering agent.

In particular, agents such as phenylacetate or phenylbutyrate compoundsare preferred, which act to remove glutamine from the circulation(glutamine being formed by the action of GS). The second or furtheragent, such as an ammonia lowering agent, may be administeredseparately, sequentially or simultaneously with the GS protein,including in the same formulation or composition, or in a separatecomposition or formulation.

Accordingly, in a further aspect, the invention provides a productcomprising a GS protein for systemic non-oral administration and afurther therapeutic agent, as a combined preparation for separate,simultaneous or sequential use in treating or preventing hyperammonemia.

The second or further agent may be administered by the sameadministration route or by a different administration route, includingorally. Thus, in one exemplary embodiment the second or further, such asan ammonia lowering agent, agent may be administered orally, or by othersystemic means and the GS may be administered by non-oral systemicmeans.

Ammonia lowering agents, such as nitrogen scavengers (including sodiumphenylacetate, ornithine phenylacetate, sodium phenyl butyrate, orsodium benzoate) may be administered by intravenous infusion e.g. foracute management, and/or orally, e.g for long-term maintenance. The i.v.infusion may be peripheral but central i.v. infusion is preferred.Similarly amino acids such as arginine may be administered orally ori.v., for example by central i.v. infusion.

Accordingly, a yet further aspect of the present invention provides aproduct (e.g. a combination product) comprising a GS protein forsystemic non-oral administration and a further therapeutic agent as acombined preparation for separate, simultaneous or sequential use intreating or preventing hyperammonemia. In a suitable embodiment, such afurther therapeutic agent may be an ammonia lowering agent. Moresuitably the ammonia lowering agent may be a nitrogen scavenger. Moresuitably, the nitrogen scavenger may be a pharmaceutically acceptablesalt of phenylacetic acid, more suitably sodium phenylacetate.

Alternatively viewed, this aspect of the invention also provides a kitcomprising (a) a GS protein for systemic non-oral administration and (b)a further therapeutic agent. Suitably, the further therapeutic may beeffective against hyperammonemia. Suitably, the further therapeuticagent is an ammonia lowering agent, more suitably a nitrogen scavenger.

Accordingly, in a further aspect the invention provides a kit comprising(a) a GS protein for systemic non-oral administration and (b) a nitrogenscavenger. Suitably, the nitrogen scavenger may be a pharmaceuticallyacceptable salt of phenylacetic acid, more suitably sodiumphenylacetate.

Such kits may be provided for use in treating or preventinghyperammonemia. The components of the kit may be provided as separatepharmaceutical compositions comprising the agent(s) in question togetherwith one or more pharmaceutically-acceptable carriers or excipients. Thecomposition(s) of the invention can be administered once or more thanonce. If the composition(s) are administered more than one time, theycan be administered at regular intervals or as needed, for example asdetermined by a clinician. Regular intervals can include, for example,approximately daily, weekly, bi-weekly, monthly, or any other interval.Selecting a treatment protocol is well within the level of skill of theskilled artisan. For example, a protocol can be determined based uponstudies in animal models. In other example, repeat doses of thecomposition(s) can be administered to a subject if the ammonia level inthe blood, is above a predetermined level.

The use of the GS protein, or the use of the ammonia lowering agent incombination with a GS protein, according to the present invention isadvantageous for the treatment of subjects for whom gene therapy is notsuitable or appropriate, for example children or subjects who arerefractory to gene therapy. Such refractory subjects may include forexample those who have previously been exposed to, or have had an immunereaction to, the viral vector used for delivery of the gene therapy.

Advantageously, the use of a protein as the therapeutic agent allowshigher doses of the active protein to be delivered to the subject andfor doses to be adjusted according to subject and to need. Furthermore,as compared to gene therapy, protein therapy allows for a much fasterresponse, and hence is more suitable for emergency use.

As mentioned, one aspect of the invention relates to a pharmaceuticalcomposition comprising an ammonia lowering agent, for use in combinationwith GS protein for use in treating or preventing hyperammonemia. Theammonia lowering agent can be formulated as a pharmaceutical compositionfor administration to a subject. The ammonia lowering agent, can beformulated in any conventional manner by mixing a selected amount of thenitrogen scavenger, with one or more physiologically or pharmaceuticallyacceptable carriers or excipients. Suitably, the composition may be fornon-oral systemic administration, or oral administration. It will beappreciated that the pharmaceutical composition may also comprise GSprotein. In such an embodiment, the composition will be formulated fornon-oral systemic administration.

Selection of the carrier or excipient is within the skill of theadministering profession and can depend upon a number of parameters,such as the mode of administration. In some examples the ammonialowering agent, is provided as a fluid. In other instances, the ammonialowering agent, is provided in dried form. Such a dried form can berehydrated prior to administration by the addition of a suitablesolution, such as water, buffer, saline or other suitable solution. Theammonia lowering agent can be formulated for direct administration orcan be formulated for dilution or other modification. Accordingly, theammonia lowering agent can be formulated in single (or unit) dosageforms or multiple dosage forms. Examples of single dose forms includeampoules and syringes. Examples of multiple dose forms include vials andbottles that contain multiple unit doses.

The concentrations of the ammonia lowering agent in the formulations areeffective for delivery of an amount of ammonia lowering agent that, uponadministration, is effective to remove nitrogen and/or ammonium from thecirculation, or reduce or inhibit ammonia production. The concentrationsand amounts will depend on several factors, including the levels of thesubstrates in the subject, and mode of administration, and can beempirically determined. Exemplary concentrations of the ammonia loweringagent in the compositions provided herein include, but are not limitedto, concentrations of or about 0.1, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 2000,3000, 4000 or 5000 mg/mL of ammonia lowering agent or more.

To formulate the ammonia lowering agent composition, in one embodimentthe weight fraction the ammonia lowering agent is dissolved, suspended,dispersed, or otherwise mixed in a selected vehicle at the desiredconcentration. The resulting mixtures are solutions, suspensions,emulsions and other such mixtures, and can be formulated as anon-aqueous or aqueous mixture, including but not limited to, asolution, suspension, paste, gel, aerosol, spray, or any otherformulation suitable for systemic administration.

Generally, the ammonia lowering agent is prepared in view of approvalfrom a regulatory agency or otherwise prepared in accordance withgenerally recognized pharmacopeia for use in animals and in humans. Thecomposition can include carriers such as a diluent, excipient, orvehicle. Such pharmaceutical carriers can be sterile liquids, such aswater and oils. Saline solutions and aqueous dextrose and glycerolsolutions also can be employed as liquid carriers, particularly forinjectable solutions. Compositions can contain along with an activeingredient: a diluent such as lactose, sucrose, dicalcium phosphate, orcarboxymethylcellulose; a lubricant, such as magnesium stearate, calciumstearate and talc; and a binder such as starch, natural gums, such asgum acaciagelatin, glucose, molasses, polvinylpyrrolidine, cellulosesand derivatives thereof, povidone, crospovidones and other such bindersknown to those of skill in the art. Suitable pharmaceutical excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, andethanol. A phenylacetic acid, or a pharmaceutically acceptable saltthereof composition, if desired, also can contain minor amounts ofwetting or emulsifying agents, or pH buffering agents, for example,acetate, sodium citrate, cyclodextrine derivatives, sorbitanmonolaurate, triethanolamine sodium acetate, triethanolamine oleate, andother such agents. Other examples of suitable pharmaceutical carrierswill be known to those skilled in the art.

The ammonia lowering agent can be administered by any method and routethat delivers the compound to the body. In certain embodiments, theammonia lowering agent can be administered parenterally. A skilledartisan would readily understand and be able to select appropriate modesof administration or delivery, including, but not limited to, oral,intravenous, intramuscular, intradermal, transdermal, subcutaneous, orintraperitoneal administration, as well as by any combination of any twoor more thereof, formulated in a manner suitable for each route ofadministration.

As well as in pharmaceutical compositions, the ammonia lowering agentmay also be formulated or administered in other ways, for example in anutritional composition such as a dietary supplement, for example aparenteral nutrition composition (e.g. alone or alongside othersupplement ingredients). Suitably, such a composition may be for oral ornon-oral administration.

As mentioned, the ammonia lowering agent is for administration incombination with GS protein. It will be appreciated that the ammonialowering agent may be administered separately, sequentially orsimultaneously with the GS protein, including in the same formulation orcomposition, or in a separate composition or formulation. Thus, in oneexemplary embodiment the ammonia lowering agent may be administeredorally, or by other systemic means and the GS may be administered bynon-oral systemic means.

Exemplary therapeutically effective doses of the ammonia lowering agent,include but are not limited to, from about 1 mg/kg body weight per dayto or to about 2000 mg/kg body weight per day, including from or fromabout 10 mg/kg to or to about 1000 mg/kg body weight per day, or from orfrom about 100 mg/kg to or to about 500 mg/kg body weight per day. Thus,for example, a subject can be administered 1, 2, 3, 4, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or 2000 mg or more ofthe ammonia lowering agent per kg body weight per day. Other exemplarytherapeutically effective doses of the ammonia lowering agent, includebut are not limited to, from about 1 g/day to about 50 g/day. Thus, forexample, a subject can be administered 1, 2, 3, 4, 5, 10, 20, 30, 40, or50 grams, or more of the ammonia lowering agent per day. It will beappreciated that the effective dose may vary depending on the ammonialowering agent. The GS composition, and/or the composition comprisingthe ammonia lowering agent or composition comprising a further agentwhich is not an ammonia lowering agent, if desired, can be presented ina package, in a kit or dispenser device, such as a syringe with aneedle, or a vial and a syringe with a needle, which can contain one ormore unit dosage forms. The kit or dispenser device can be accompaniedby instructions for administration. In an embodiment where the GScomposition and the composition comprising the ammonia lowering agentare separate, the kit may comprise the GS composition and thecomposition comprising the ammonia lowering agent. Suitably, in such anembodiment, the kit may comprise a GS composition, and a nitrogenscavenger. Suitably, in such an embodiment, the kit may comprise a GScomposition, and a composition comprising phenylacetate, for examplesodium phenylacetate. The compositions can be packaged as articles ofmanufacture containing packaging material, the composition, and a labelthat indicates that the composition is for administration to subjectsfor the treatment of hyperammonemia or a disease or condition associatedwith hyperammonemia.

In a further aspect, the invention provides an expression vectorencoding glutamine synthetase or a biologically active fragment orvariant thereof, for use in the treatment or prevention ofhyperammonemia, wherein the vector is for systemic administration.

In a suitable embodiment, the vector further encodes an ammonia loweringagent.

In another aspect, the invention provides an expression vector encodingglutamine synthetase or a biologically active fragment or variantthereof, for use in combination with an ammonia lowering agent, for usein the treatment or prevention of hyperammonemia.

In another aspect, the invention provides an ammonia lowering agent foruse in combination with an expression vector encoding glutaminesynthetase or a biologically active fragment or variant thereof, for usein the treatment or prevention of hyperammonemia.

In another aspect, the invention provides a cell comprising anexpression vector selected from the group consisting of: an expressionvector encoding glutamine synthetase or a biologically active fragmentor variant thereof, for use in the treatment of hyperammonemia, and anexpression vector encoding glutamine synthetase or a biologically activefragment or variant thereof, for use in combination with an ammonialowering agent, for use in the treatment or prevention ofhyperammonemia.

In another aspect, the invention provides use of an expression vectorencoding glutamine synthetase or a biologically active fragment orvariant thereof, for the manufacture of a composition for the treatmentor prevention of hyperammonemia, wherein the composition is for systemicadministration.

In another aspect, the invention provides use of an expression vectorencoding glutamine synthetase or a biologically active fragment orvariant thereof, for the manufacture of a composition, for use incombination with an ammonia lowering agent, for the treatment orprevention of hyperammonemia.

In another aspect, the invention provides use of an ammonia loweringagent for the manufacture of a composition, for use in combination withan expression vector encoding glutamine synthetase or a biologicallyactive fragment or variant thereof, for use in the treatment orprevention of hyperammonemia.

In another aspect, the invention provides a method of treating orpreventing hyperammonemia in a subject, said method comprising systemicadministration of an expression vector encoding glutamine synthetase ora biologically active fragment or variant thereof.

In another aspect, the invention provides a method of treating orpreventing hyperammonemia in a subject, said method comprisingadministration of an expression vector encoding glutamine synthetase ora biologically active fragment or variant thereof, and an ammonialowering agent.

In another aspect, the invention provides a composition comprising anexpression vector encoding glutamine synthetase or a biologically activefragment or variant thereof, for use in the treatment or prevention ofhyperammonemia, wherein the composition is for systemic administration.

In a suitable embodiment, the composition may further comprise anammonia lowering agent.

In another aspect, the invention provides a composition comprising anexpression vector encoding glutamine synthetase or a biologically activefragment or variant thereof, for use in combination with an ammonialowering agent, for use in the treatment or prevention ofhyperammonemia.

In another aspect, the invention provides a composition comprising anammonia lowering agent, for use in combination with an expression vectorencoding glutamine synthetase or a biologically active fragment orvariant thereof, for use in the treatment or prevention ofhyperammonemia.

In another aspect, the invention provides a kit comprising a with anexpression vector encoding glutamine synthetase or a biologically activefragment or variant thereof, and a further therapeutic agent. Suitably,the further therapeutic agent may be an agent that is effective againsthyperammonemia. Suitably the agent that is effective againsthyperammonemia is an ammonia lowering agent or an amino acid or ureacycle intermediate thereof.

In another aspect, the invention provides a product comprising anexpression vector encoding glutamine synthetase or a biologically activefragment or variant thereof and a further therapeutic agent, as acombined preparation for separate, simultaneous or sequential use intreating or preventing hyperammonemia. Suitably, the further therapeuticagent may be an agent that is effective against hyperammonemia. Suitablythe agent that is effective against hyperammonemia is an ammonialowering agent or an amino acid or urea cycle intermediate thereof. Moresuitably, the further therapeutic agent is a nitrogen scavenger.

In a suitable embodiment, the expression vector may be viral ornon-viral. By way of example, a suitable viral expression vector may bederived from a virus selected from the group consisting ofparamyxovirus, retrovirus, adenovirus, lentivirus, pox virus,alphavirus, and herpes virus. Other suitable viral vectors will be knownto those skilled in the art.

Suitable non-viral expression vectors may be selected from the groupconsisting of inorganic particle expression vectors (such as calciumphosphate, silica, and gold), lipid based particle expression vectors(for example cationic lipids, lipid nano emulsions, and solid lipidnanoparticles) and polymer based particle expression vectors (forexample peptides, polyethylenimine, chitosan, and dendimers). Othersuitable non-viral expression vectors will be known to those skilled inthe art.

Suitable systemic administration methods will be known to those skilledin the art. By way of example, systemic administration may be achievedby parenteral route of administration, such as intravenous orsubcutaneous route. It will be appreciated that “systemicadministration” allowed the product (such as glutamine synthetase or abiologically fragment thereof) of an expression vector to be expressedwithin multiple sites in the patient. In the context of the presentinvention, systemic administration does not include intramuscularadministration.

The term “biologically active” refers to a fragment or variant of thenucleic acid encoding glutamine synthetase (SEQ ID NO: 1) exhibiting theability to convert glutamate to glutamine. The protein according to SEQID NO: 1 is encoded by the nucleic acid sequence according to SEQ ID NO:2. Thus, it will be appreciated that the vector, may comprise a fragmentor variant of SEQ ID NO:2 which encodes a biologically active fragmentor variant of glutamine synthetase.

The term “variant” as used herein refers to a polypeptide comprising analteration of the primary structure of the polypeptide of SEQ ID NO: 1.Suitably, a variant may share 70% or more identity with the polypeptideof SEQ ID NO: 1; 80% or more identity with the polypeptide of SEQ ID NO:1; 90% or more identity with the polypeptide of SEQ ID NO: 1; 95% ormore identity with the polypeptide of SEQ ID NO: 1; 96% or more identitywith the polypeptide of SEQ ID NO: 1; 97% or more identity with thepolypeptide of SEQ ID NO: 1; 98% or more identity with the polypeptideof SEQ ID NO: 1; or even 99% or more identity with polypeptide of SEQ IDNO: 1. A variant may differ from the polypeptide of SEQ ID NO: 1 by 1%or more, 2% or more, 3% or more, 4% or more, 5% or more, 10% or more,20% or more, or even 30% or more with reference to sequence according toSEQ ID NO: 1.

The term “fragment” as used herein refers to a polypeptide comprising analteration to the length of the primary structure of the polypeptide ofSEQ ID NO: 1. A suitable fragment may comprise at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% of the full length of SEQ ID NO: 1. Indeed, asuitable variant may comprise at least 96%, at least 97%, at least 98%,or at least 99% of SEQ ID NO: 1.

It will be appreciated that (except where the context requiresotherwise), embodiments described with reference to the GS protein foruse in preventing hyperammonemia, the GS protein for use in combinationwith an ammonia lowering agent, ammonia lowering agent for use incombination with a GS protein, their uses, methods of treatment,compositions, kit and product, will generally be applicable, to theremaining aspects of the invention.

The present invention will now be further described with reference tothe following non-limiting Examples and Figures in which:

FIG. 1 shows the results of size exclusion chromatography (SEC) on aSuperose 12 column of 20 kDa N-terminal aldehyde PEG conjugates of humanGS protein as prepared in Example 1. The graph shows that multimers wereeluted in fractions 8 and 9, and monomer in fraction 10;

FIG. 2 shows a comparison of the in-vitro GS activity of various GScandidates: PEG-conjugated variants (Trin GS1, Trin GS2, Trin GS3, TrinGS 4) versus non-conjugated GS (wt GS) and negative control. GlutamineSynthetase activity is shown as OD 570 nm according to the assay ofAcosta et al., 2009, World J. Gastroenterol., 15(23), 2893-2899. TrinGS1—(N-term Ald monomer); Trin GS2—Nof-20; Trin GS3—Nof-30, TrinGS4—N-term Ald multimer;

FIG. 3 shows PEG ELISA results on plasma pre- and post-dosing of male,wild-type (wt) CD1 mice of various conjugates at (A) baseline, (B) 24hours post-dose and (C) 72 hours post-dose. (Trin1—N-terminal Aldehydeconjugated GS monomer; Trin2—Nof-20 GS conjugated multimer; Trin3—Nof-30 conjugated GS multimer; Trin4—N-terminal Aldehyde conjugatedPEG multimer);

FIG. 4 shows GS activity (OD 535 nm) results in liver lysates in dosedwtCD1 mice at 2.5 mg/kg, 3 days post-dosing, as described in Example 3.

FIG. 5 A shows liver GS activity assay results. FIG. 5 B shows bloodplasma GS activity assay results. Both, liver and blood plasma GSactivity was assayed in BDL rats treated with GS protein, and GS proteinwith nitrogen scavenger.

FIG. 6 illustrates ammonia blood levels measured in BDL rats treatedwith GS protein, and GS protein with nitrogen scavenger.

FIG. 7 illustrates a graph showing percentage of oedema in theprefrontal cortex in BDL rats treated with GS protein, or GS proteinwith nitrogen scavenger.

FIG. 8 shows the results of a rotarod grip test in BDL rats treated withGS protein, or GS protein with nitrogen scavenger.

FIG. 9 a graph showing ammonia levels in OTC mice treated with GSprotein, or GS protein with nitrogen scavenger (SP— sodiumphenylacetate).

FIG. 10 shows the results of ammonia levels in plasma and liver GSactivity in OTC mice treated with GS protein or DS protein with nitrogenscavenger (SP—sodium phenylacetate)

EXAMPLE 1 Production and Purification of GS Protein and GS Protein-PEGConjugates

Production of human glutamine synthetase (GS): pET30a+ vector,containing the gene for human GS (SEQ ID NO. 5 comprising a 5′ sequenceencoding a His-tag and the linker GGGGS at the N-terminal end of the GSand codon optimised for expression in bacteria) was used in an E. coliexpression system. After plasmid construction, evaluation for theexpression of GS was performed with a wide range of induction (IPTG) andexpression temperatures. Human GS was solubly expressed in the constructas detected by SDS-PAGE. Lysis buffer (50 mM Tris pH 8.0, 10% glycerol,0.1% Triton X-100, 100 ug/ml lysozyme, 1 mM PMSF, 3 Units DNAse, 2 mMMgCl) was used to extract soluble protein from cells. Soluble proteinwas extracted following centrifugation. After expression studies, thebest condition found with BL21 (DE3) cells, cultured and induced with0.1 mM IPTG at 25° C. for 16 hours. Other conditions tried, includedusing varied IPTG induction (from 0.01M-0.1M IPTG), various incubationtemperatures (ranging from 16° C.-37°), and induction incubation timesfrom 4-16 hours.

Purification of the expressed GS: the first step purification of theexpressed protein comprised His tag purification with Ni-NTA beads,washing with 20 mM Imidazole, and elution with 300 mM Imidazole.

Protein PEG conjugation: the GS protein was conjugated under reducingconditions (with the use of 20 mM Sodium Cyano borohydride) toN-terminal aldehyde 20 kDa peg for 16 hours (Dr Reddy's 20 kDaN-terminal Aldehyde PEG).

Final purification: Conjugated protein was further purified using SECchromatography. A Superose 6 or Superose 12 column (see FIG. 1) wasused. Multimers were found in fractions 8+9. Fraction 10 in Superose 12comprised (dilute) multimer. In Superose 6, multimers were found infractions 8+9 and monomer in fractions 12/13.

A final formulation of the GS in PBS, pH 7.4 containing trehalose andsucrose was prepared.

EXAMPLE 2 Activity of GS Preparations

Various GS preparations and PEG conjugates prepared according to Example1 were tested for GS activity using the assay of Acosta et al., 2009(supra), modified from the original assay described in Ehrenfeld et al.,1963, J. Biol. Chem. 238(11), 3711-3716.

100 ug of purified protein sample was added to the following reactionbuffer: 150 μL stock solution (100 mmol/L imidazole-HCl buffer [pH7.1],40 mmol/L MgCl2, 50 mmol/L, β-mercaptoethanol, 20 mmol/L ATP, 100mmol/L, glutamate and 200 mmol/L hydroxylamine, adjusted to pH 7.2)Tubes were incubated at 37° C. for 15 min. The reaction was stopped byadding 0.6 mL [2× concentration] ferric chloride reagent (0.37 mol/LFeCl3, 0.67 mol/L HCl and 0.20 mol/L trichioroacetic acid). Samples wereplaced for 5 minutes on ice. Precipitated proteins were removed bycentrifugation at 10,000 g, and the absorbance of the supernatants wasread at 535-570 nm against a reagent blank. The results are shown inFIG. 2. Trin1—(N-term Aldehyde monomer PEG of 20 kD size, obtained fromDr Reddy's); Trin2—Nof-20 conjugated GS, which was conjugated to the GSprotein with a monofunctional linear 20 kD PEG, NHS active ester,obtained from NOF corporation); Trin 3—Nof-30, conjugated to the GSprotein with a monofunctional linear 30 kD PEG, NHS active ester,obtained from NOF corporation) Trin4—N-term Ald, GS multimers). Trin 4(N-term Ald multimer) showed the best activity, with a very similaractivity profile compared to the wt GS (non-conjugated), though activityof other conjugates was similar.

EXAMPLE 3 Dosing of GS Protein to Mice—Effects on Plasma Levels of GSProtein-PEG Conjugates

Male, wild-type (wt) CD1 mice were dosed at 2.5 mg/kg with subcutaneous(s.c) dosing of various GS protein and PEG conjugates prepared asdescribed in Example 1 (Trin1—N-terminal Aldehyde conjugated GS monomer;Trin2—Nof-20 GS conjugated multimer; Trin 3—Nof-30 conjugated GSmultimer; Trin4—N-terminal Aldehyde conjugated PEG multimer). The ELISAwas conducted according to the protocol outlined by the manufacturer(Abcam PEG ELISA kit, ab133065). Results of the plasma ELISA, as shownin FIG. 3, show either very low or undetectable levels for unconjugatedwt GS, as expected at all timepoints. After 24 hours, several candidateswere found to be at a high level in plasma; however, after 72 hourspost-dosing, Trin-GS 4 (N-terminal Aldehyde conjugated PEG GS multimer)showed the highest presence. N=2 animals in each group. Thus, thisexperiment shows that systemic administration of GS protein may be usedsuccessfully to obtain high circulating levels of the GS PEG conjugates,and in particular at levels which may be therapeutically effective oractive.

EXAMPLE 4 Dosing of GS Protein to Mice—GS Activity Levels of LiverLysates

The activity assay was performed as described in Example 2, with theexception that 500 μg of liver lysate (from culled mice from theexperiment of Example 3) was added to each reaction where appropriate.The results are shown in FIG. 4. The GS activity results in liverlysates 3 days post-dosing demonstrate that the superior candidate wasthe N-terminal aldehyde conjugated PEG GS multimer, which was the onlycandidate to show significant activity above baseline compared to thevehicle (saline-dosed) control. N=2 animals in each group.

EXAMPLE 5

The Otc^(spf-ash) Mouse model of urea cycle disorder (OTC deficiency)was used to show the effects of GS and GS+SP. The details of the miceused can be found at https://www.jax.org/strain/001811 (B6EiC3Sna/A-Otc^(spf-ash)/J). They are fed normal chow. The ages were variablefrom about 10 weeks to 23 weeks, with groups well-matched. All animalsare male hemizygous (as OTC is X-linked, it is present only on the Xchromosome of the males, therefore the mice are knockout).

All groups (vehicle, GS and GS+SP; where GS=Glutamine synthetase,GS+SP=Glutamine Synthetase+Sodium Phenlyacetate) were treated asfollows: The experiment ran from a Tuesday until the following Wednesday(8 days). SP was dosed i.p. 350 mg/kg twice daily; GS was dosed in alltreated groups for the first 4 days (i.p. @ 40 mg/kg once daily), then abreak of 2 days [a weekend], and 3 more days of dosing with GS @ 40mg/kg i.p.

Mice were culled on day 8, and blood extracted, spun down for plasma,and this plasma was used for ammonia quantitation (see method below).

Genotyping is performed using standard methods described in theliterature.

Materials and Methods

All experiments were performed in accordance with the Animals(Scientific Procedures) Act of 1986, which was revised according to theEuropean Directive2010/63/EU. All animals received humane care accordingto the criteria outlined in the Guide for the Care and Use of LaboratoryAnimals (National Institutes of Health publication 86-23; revised 1985).All the animals used in these experiments were Male Sprague-Dawley rats(body weight, 250 g at the beginning of the experiments) were obtainedfrom Charles River Laboratories (Kent, UK) and divided into 5 groups:bile duct ligated animals+ammonia+saline serum (BDL+HA+SS, n=6), bileduct ligated animals+ammonia+sodium phenylacetate (BDL+HA+SP, n=6), bileduct ligated animals+ammonia+sodium phenylacetate+glutamine synthetase(BDL+HA+SP+GS, n=5), bile duct ligated animals+ammonia+glutaminesynthetase (BDL+HA+GS, n=6), sham-operated animals+glutamine synthetase(SHAM+GS, n=5). Treatment comprising SP and GS may be referred to as“COMBO”.

Bile Duct Ligation Surgery

Under general anesthesia (5% isoflurane in 100% oxygen for induction, 2%isofluorane in air for maintenance) rats underwent triple ligation ofthe bile duct (way of a small laparotomy) to induce chronic liver injuryand were studied 28 days after surgery. A midline abdominal incision wasmade under anesthesia. In the BDL group, the common bile duct wasisolated, triply ligated with 3-0 silk, and sectioned between theligatures. The sham-operated group performed the same procedure withoutthe sectioning between the ligatures. After BDL all animals continued togain weight and were comparable with sham controls. The overallmortality in both groups was less than 10% and occurred within 36 hoursof the operation.

Noncirrhotic Hyperammonemia Condition

Twenty-three rats were administered a hyperammonemic (HA) diet. Theamino acid recipe used for a stock of approximately 100 g was: 15 gleucine, 7.7 g phenylalanine, 7 g glutamate, 10 g alanine, 4.4 gproline, 5.8 g threonine, 11 g aspartate, 5 g serine, 4.8 g glycine, 3.3g arginine, 9.6 g lysine, 8.4 g histidine, 3 g tyrosine, 1.5 gtryptophan, and 10.6 g valine. 25 g of this mix (mixed 1:5 with standardrodent chow powder) was freshly prepared daily and rats were given freeaccess to it for 5 days. The recipe approximates the amino acidcomposition of a rodent haemoglobin, [1] mimicking the effect ofgastrointestinal bleeding, which is known to result in systemichyperammonemia [2].

Sodium Phenylacetate Condition

Eleven rats were administered a sodium phenylacetate (SP) diet. 0.3 g/kga day for 5 days was mixed with the chow powder and freshly prepareddaily.

Glutamine synthetase Condition

Sixteen rats were injected with GS intraperitoneally every two days (day1 and day 3). The total volume injected was 3 mls i.p., which allows for18-22 mg/kg of GS.

Blood Sampling and Biochemistry

Plasma samples were collected from the leg vein at different timepointsin all groups. The timepoints were counted after the treatment withglutamine synthetase as follows: 6 hours, 24 hours, 48 hours and 5 days.Analyses were conducted for plasma ammonia levels in every timepointusing 200 μl of respective plasma using a Cobas Integra 400multi-analyser with the appropriate kits (Roche-diagnostics, BurgessHill, West Sussex, UK).

Brain Edema

This was measured using the dry weight technique as described previously[3, 4]. Briefly, oven dried Eppendorf's were weighed with a sensitiveelectronic scale, then prefrontal cortex, striatum, hippocampus,cerebellum and cortices of each animal were placed into eachrespectively labelled Eppendorf and reweighed; all samples where within0.1 mg difference. The dry weight was determined after Eppendorf'sloaded with individual brain samples were dried in an oven at 60° C. for7 days. Tissue water content was then calculated as % H2O=(1−dry wt/wetwt)×100%.

Test for Assessment Locomotor Activity: RotaRod-Accelerod Test

This test of motor performance consists of a motor-driven rotating rodthat enables us to assess motor coordination and resistance to fatigue(Jones and Roberts 1968). The accelerating rotarod 7750 of Ugo Basile(Ugo Basile Biological Research Apparatus, Italy) was used for the rats.The procedure followed has two parts. In the first one, the animals wereplaced in the apparatus and the speed was maintained constant at 2 rpmfor 60 s. In the second part, the rats were evaluated for 5 min in theaccelerod test session, in which the rotation rate constantly increaseduntil it reached 20 rpm. Latency to fall off the rod and the actualrotation speed were recorded in the pre- and post-treatment conditionsfor all groups after 1 hour treatment.

Ammonia Determination in Blood Using the TCA Direct Method

The method described in the paper (Clin Chim Acta. 1968 October;22(2)183-86) was used to measure plasma ammonia concentration, asfollows.

Principle

In an alkaline solution ammonium ions react with hypochlorite to formmonochloramine. In the presence of phenol and an excess of hypochlorite,the monochloramine will form a blue coloured compound, indophenol, whennitroprusside is used as a catalyst. The concentration of ammonium isdetermined spectrophotometrically at 630 nm.

Method

Dissolve 3.5 g of phenol and 0.04 g sodium nitroprusside in 100 mldistilled water to prepare reagent A.

Dissolve 1.8 g sodium hydroxide in 48 mls in distilled water and add 4mls of 1M sodium hypochlorite solution to prepare reagent B.

Add 150 μl of 5% TCA to 50 μl to each plasma sample and centrifuge at10,000 RPM at 4° c. for 10 minutes. Take 50 μl of the supernatant andput in 96 well plate to which is added 50 μl of both reagents A and B.

Standard ammonium chloride concentrations for the calibration curve aremade by dissolving ammonium chloride in distilled water and seriallydiluting to make concentrations ranging from 400 μmol to 3 μmol.Distilled water is used as the blank The well plate is covered fromlight and incubated at 50° c. for 60 minutes. Absorbance is measured at630 nm using a spectrophotometer to determine the ammonia concentration.

Results Dosing of GS Protein to Mice—GS Activity Levels in Liver andBlood

The activity assay was performed as described in the materials andmethods section above. The results are shown in FIGS. 5A and B. Theresults in rat liver measured at day 5, show that GS activity is best inthe SHAM+GS group. Additionally, it can been seen from FIG. 5A that GSand GS+SP treatment increase GS activity in the livers of mice whichhave undergone BDL. When measured in blood, the results show that GSactivity is best in the BDL+GS group. Additionally, from FIG. 5B, it canbe seen that GS activity in blood is consistant over time, even 24 hrsand 48 hrs post dosing.

Dosing of GS Protein to Mice—Ammonia Concentrations in the BDL Rat

As seen in FIG. 6, ammonia levels are highest in the BDL rat. Treatmentwith GS, GS+SP, and SP, each resulted in a significant reduction ofammonia levels in the blood. GS reduced ammonia levels following 2doses. Treatment with GS+SP reduced the ammonia levels mostsignificantly, suggesting a synergistic effect.

Dosing of GS Protein to Mice—Brain Swelling in the BDL Rat

Brain oedema was measured in the prefrontal cortex. Treatment with GSwas found to reduce brain oedema most significantly compared totreatment to with SP, and even treatment with SP+GC (FIG. 7). Treatmentwith SP did not statistically significantly reduce the swelling ascompared to the control (i.e. BDL mice without treatment).

Dosing of GS Protein to Mice—Brain and Physical Function in the BDL Rat

FIG. 8 shows the results of a rotarod grip test. Surprisingly, GS dosingwas found to improve performance in all tested mice groups. Treatmentwith SP alone did not lead to statistically significant effects, buttreatment with GS+SP shows the best improvement, suggesting a synergisiceffect.

Treatment of Mice with OTC Deficiency

As shown in FIG. 9, ammonia is very significantly decreased in thetreated groups.

In FIG. 10, it is seen that in the OTC mice treated with GS or GS & SP,plasma GS activity increased from 0.2 in the vehicle group, to 0.8 in GSonly group and ˜1.1 in the GS & SP group. Liver GS activity increasedfrom ˜0.175 in the vehicle group, to ˜2.8 in GS only group and ˜2.5 inthe GS & SP group

SUMMARY OF RESULTS

In summary, the administered GS is biocompatible, safe and improvesblood and liver GS activity. It also leads to a reduction in ammonia andbrain oedema, as well as improves neurocognitive and/or physicalfunction. Additionally, the data suggests that treatment with SP and GSmay have a synergstic effect.

REFERENCES

-   [1] Riggs A. The amino acid composition of some mammalian    hemoglobins: mouse, guinea pig, and elephant. J Biol Chem 1963;    238:2983-2987.-   [2] Balata S, Olde Damink S W, Ferguson K, Marshall I, Hayes P C,    Deutz N E, Williams R, Wardlaw J, Jalan R. Induced hyperammonemia    alters neuropsychology, brain M R spectroscopy and magnetization    transfer in cirrhosis. Hepatology 2003; 37:931-939.-   [3] Stewart-Wallace A M. A biochemical study of cerbral tissue, and    of changes in cerebral oedema. Brain 1939; 62: 426-38.-   [4] Traber P G, Ganger D R, Blei A T. Brain edema in rabbits with    galactosamine-induced fulminant hepatitis. Regional differences and    effects on intracranial pressure. Gastroenterology 1986; 91:    1347-56.

SEQUENCES: SEQ ID NO. 1 [Full human protein]MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEWNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRACLYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIR TCLLNETGDEPFQYKNSEQ ID. NO. 2 (ONLY Methionine is cleaved for themature protein in vivo):TTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEWNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRACLYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRT CLLNETGDEPFQYKNSEQ ID NO. 3 cDNA CGAGAGTGGGAGAAGAGCGGAGCGTGTGAGCAGTACTGCGGCCTCCTCTCCTCTCCTAACCTGCTCTCGCGGCCTACCTTTACCCGCCCGCCTGCTCGGCGACCAGAACACCTTCCACCATGACCACCTCAGCAAGTTCCCACTTAAATAAAGGCATCAAGCAGGTGTACATGTCCCTGCCTCAGGGTGAGAAAGTCCAGGCCATGTATATCTGGATCGATGGTACTGGAGAAGGACTGCGCTGCAAGACCCGGACCCTGGACAGTGAGCCCAAGTGTGTGGAAGAGTTGCCTGAGTGGAATTTCGATGGCTCCAGTACTTTACAGTCTGAGGGTTCCAACAGTGACATGTATCTCGTGCCTGCTGCCATGTTTCGGGACCCCTTCCGTAAGGACCCTAACAAGCTGGTGTTATGTGAAGTTTTCAAGTACAATCGAAGGCCTGCAGAGACCAATTTGAGGCACACCTGTAAACGGATAATGGACATGGTGAGCAACCAGCACCCCTGGTTTGGCATGGAGCAGGAGTATACCCTCATGGGGACAGATGGGCACCCCTTTGGTTGGCCTTCCAACGGCTTCCCAGGGCCCCAGGGTCCATATTACTGTGGTGTGGGAGCAGACAGAGCCTATGGCAGGGACATCGTGGAGGCCCATTACCGGGCCTGCTTGTATGCTGGAGTCAAGATTGCGGGGACTAATGCCGAGGTCATGCCTGCCCAGTGGGAATTTCAGATTGGACCTTGTGAAGGAATCAGCATGGGAGATCATCTCTGGGTGGCCCGTTTCATCTTGCATCGTGTGTGTGAAGACTTTGGAGTGATAGCAACCTTTGATCCTAAGCCCATTCCTGGGAACTGGAATGGTGCAGGCTGCCATACCAACTTCAGCACCAAGGCCATGCGGGAGGAGAATGGTCTGAAGTACATCGAGGAGGCCATTGAGAAACTAAGCAAGCGGCACCAGTACCACATCCGTGCCTATGATCCCAAGGGAGGCCTGGACAATGCCCGACGTCTAACTGGATTCCATGAAACCTCCAACATCAACGACTTTTCTGGTGGTGTAGCCAATCGTAGCGCCAGCATACGCATTCCCCGGACTGTTGGCCAGGAGAAGAAGGGTTACTTTGAAGATCGTCGCCCCTCTGCCAACTGCGACCCCTTTTCGGTGACAGAAGCCCTCATCCGCACGTGTCTTCTCAATGAAACCGGCGATGAGCCCTTCCAGTACAAAAATTAAGTGGACTAGACCTCCAGCTGTTGAGCCCCTCCTAGTTCTTCATCCCACTCCAACTCTTCCCCCTCTCCCAGTTGTCCCGATTGTAACTCAAAGGGTGGAATATCAAGGTCGTTTTTTTTCATTCCSEQ ID NO. 4: GS protein grown in bacteria, used in Example 1MGSSHHHHHHGGGGSMTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEWNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRACLYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETG DEPFQYKNSEQ ID NO. 5 cDNA (bacterial optimised cDNA used in Example 1).ATGGGCAGCAGCCACCACCATCACCACCACGGCGGCGGCGGTAGCATGACCACCTCGGCAAGCAGCCACCTGAATAAAGGCATCAAACAGGTGTATATGTCTCTGCCGCAGGGTGAAAAAGTTCAAGCCATGTACATTTGGATCGATGGCACCGGTGAAGGCCTGCGTTGCAAAACCCGCACGCTGGACTCAGAACCGAAATGTGTGGAAGAACTGCCGGAATGGAACTTTGATGGTAGCTCTACGCTGCAGTCGGAAGGCAGTAATTCCGACATGTATCTGGTTCCGGCGGCCATGTTTCGTGATCCGTTCCGCAAAGACCCGAACAAACTGGTGCTGTGCGAAGTTTTTAAATACAACCGTCGCCCGGCGGAAACCAATCTGCGTCATACGTGTAAACGCATTATGGATATGGTCAGCAACCAGCACCCGTGGTTCGGTATGGAACAAGAATATACCCTGATGGGTACGGATGGCCATCCGTTTGGTTGGCCGAGCAATGGTTTCCCGGGTCCGCAGGGTCCGTATTACTGCGGTGTCGGCGCAGATCGTGCTTACGGTCGCGACATTGTGGAAGCACACTATCGTGCTTGTCTGTACGCGGGTGTTAAAATCGCCGGCACCAATGCAGAAGTCATGCCGGCTCAGTGGGAATTTCAAATTGGCCCGTGCGAAGGTATCAGCATGGGCGATCATCTGTGGGTTGCTCGTTTCATCCTGCACCGCGTCTGTGAAGATTTTGGTGTGATTGCGACCTTCGACCCGAAACCGATCCCGGGCAACTGGAATGGTGCTGGCTGCCATACCAACTTTAGCACGAAAGCGATGCGTGAAGAAAATGGCCTGAAATACATCGAAGAAGCAATCGAAAAACTGTCTAAACGTCATCAGTATCACATTCGCGCCTACGATCCGAAAGGCGGTCTGGACAACGCACGTCGCCTGACCGGTTTTCACGAAACGAGCAACATCAATGATTTCTCTGCGGGCGTTGCCAATCGCTCAGCCTCGATTCGTATCCCGCGCACCGTCGGTCAAGAGAAAAAAGGCTATTTTGAAGATCGTCGCCCGAGTGCAAACTGTGACCCGTTCTCCGTGACGGAAGCCCTGATCCGCACCTGTCTGCTGAATGAAACCGGCGATGAACCGTTCCAATACAAAAAT SEQ ID NO. 6 [Lactobacillus acidophilusstrain 30SC GS] >tr|F0TG87|F0TG87_LACA3 Glutamine synthetaseOS = Lactobacillus acidophilus (strain 30SC)MSKQYTTEEIRKEVADKDVRFLRLCFTDINGTEKAVEVPTSQLDKVLTNDIRFDGSSIDGFVRLEESDMVLYPDFSTWSVLPWGDEHGGKIGRLICSVHMTDGKPFAGDPRNNLKRVLGEMKEAGFDTFDIGFEMEFHLFKLDENGNWTTEVPDHASYFDMTSDDEGARCRREIVETLEEIGFEVEAAHHEVGDGQQEIDFRFDDALTTADRCQTFKMVARHIARKHGLFATFMAKPVEGQAGNGMHNNMSLFKNKHNVFYDKDGEFHLSNTALYFLNGILEHARAITAIGNPTVNSYKRLIPGFEAPVYIAWAAKNRSPLVRIPSAGEINTRLEMRSADPTANPYLLLAACLTAGLKGIKEQKMPMKPVEENIFEMTEEERAEHGIKPLPTTLHNAIKAFKEDDLIKSALGEHLTHSFIESKELEWSKYSQSVSDWERQRYMNWSEQ ID NO. 7 [Zea Mays GS] (corn/Maize GS) >tr|B4G1P1|B4G1P1_MAIZE Glutamine synthetaseMACLTDLVNLNLSDNTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDCYTPAGEPIPTNKRYNAAKIFSSPEVAAEEPWYGIEQEYTLLQKDTNWPLGWPIGGFPGPQGPYYCGIGAEKSFGRDIVDAHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISSGDQVWVARYILERITEIAGVVVTFDPKPIPGDWNGAGAHTNYSTESMRKEGGYEVIKAAIEKLKLRHREHIAAYGEGNERRLTGRHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYVVTSMIAETTIIWKP

1-30. (canceled)
 31. A method of treating or preventing hyperammonemiain a subject, said method comprising (a) systemically and non-orallyadministering a glutamine synthetase (GSA protein to said subject, (b)systemically administering an expression vector encoding GS or abiologically active fragment or variant thereof to said subject vianon-intramuscular administration, or (c) systemically and non-orallyadministering a GS protein to said subject and administering an ammonialowering agent.
 32. A method of claim 31, wherein the GS protein is notadministered to muscle.
 33. A method of claim 31, wherein thehyperammonemia arises due to a urea cycle disorder (UCD) and/orglutamine synthetase deficiency, or is associated with organ damage,infection or failure, or arises due to non-alcoholic fatty liverdisease, acute liver failure, liver cirrhosis, renal dysfunction and/orfailure, acidaemia, fatty acid oxidation disorder, mitochondrialdisorder, or systemic infection.
 34. A method of claim 31, wherein theGS protein comprises an amino acid sequence that is at least 50%identical to the amino acid sequence set forth in SEQ ID NO: 1, or is anenzymatically-active fragment thereof.
 35. A method of claim 32, whereinthe ammonia lowering agent is selected from the group consisting of anitrogen scavenger, an ion exchange resin, an ammonia absorber, anengineered microbiome that removes ammonia, Rifaximin and Lactulose. 36.A method of claim 35, wherein the nitrogen scavenger is selected fromthe group consisting of: a pharmaceutically acceptable salt ofphenylacetic acid or a pharmaceutically acceptable pro-drug thereof, apharmaceutically acceptable salt of phenylbutyric acid or apharmaceutically acceptable pro-drug thereof, glycerol phenylbutyrate ora pharmaceutically acceptable pro-drug thereof, a pharmaceuticallyacceptable salt of benzoic acid or a pharmaceutically acceptablepro-drug thereof, and ammonia binding resin. 37-49. (canceled)
 50. A kitcomprising (a) a GS protein for systemic non-oral administration and afurther therapeutic agent or (b) an expression vector encoding a GSprotein or a biologically active fragment or variant thereof, and afurther therapeutic agent.
 51. (canceled)
 52. The kit of claim 50,wherein the further therapeutic agent is an ammonia lowering agent or anamino acid or urea cycle intermediate or analogue thereof. 53.(canceled)
 54. The kit of claim 52, wherein the ammonia lowering agentis a nitrogen scavenger selected from a group consisting of: apharmaceutically acceptable salt of phenylacetic acid or apharmaceutically acceptable pro-drug thereof, a pharmaceuticallyacceptable salt of phenylbutyric acid or a pharmaceutically acceptablepro-drug thereof, glycerol phenylbutyrate or a pharmaceuticallyacceptable pro-drug thereof, a pharmaceutically acceptable salt ofbenzoic acid or a pharmaceutically acceptable pro-drug thereof, andammonia binding resin.
 55. (canceled)
 56. The kit according to claim 50,wherein the GS protein comprises an amino acid sequence that is at least50% identical to the amino acid sequence set forth in SEQ ID NO: 1, oris an enzymatically-active fragment thereof. 57-71. (canceled)
 72. Amethod of treating or preventing hyperammonemia in a subject accordingto claim 31, comprising systemically administering an expression vectorencoding GS or a biologically active fragment or variant thereof to saidsubject and further comprising administration of an ammonia loweringagent. 73-81. (canceled)
 82. The method of claim 31, wherein the GSprotein is administered in the form of a pharmaceutical composition, ora parenteral nutrition composition.
 83. The method of claim 31, whereinthe GS protein is linked to a moiety selected from a protein, a peptide,a non-protein polymer or an affinity tag.
 84. The method of claim 83,wherein the moiety is polyethylene glycol (PEG) and is linked to the GSprotein at an N terminus of the GS protein.
 85. The method of claim 83,wherein the GS protein is linked to the moiety via a peptide linker or achemical linkage, or via a covalent bond.
 86. The method of claim 31,comprising parenteral or subcutaneous administration to the subject. 87.The method of claim 31, wherein the GS protein is provided as apreparation comprising multimeric forms of the protein or the GS proteinis provided in monomeric form.
 88. A glutamine synthetase proteinconjugated to polyethylene glycol (PEG).
 89. A glutamine synthetaseprotein conjugated to PEG according to claim 88, wherein the PEG isN-terminal aldehyde PEG.
 90. A glutamine synthetase protein conjugatedto an N-terminal amino acid or polypeptide sequence.
 91. A glutaminesynthetase protein according to claim 90, wherein the protein furthercomprises PEG, wherein PEG is conjugated to the N-terminal sequence. 92.A glutamine synthetase protein according to claim 91 wherein PEG isN-terminal aldehyde PEG.
 93. A glutamine synthetase protein according toclaim 90 wherein the N-terminal amino acid sequence conjugated toglutamine synthetase is about 14 amino acids in length.